Two-Photon Autofluorescence Imaging of Fixed Tissues: Feasibility and Potential Values for Biomedical Applications.

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50 µm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750 nm, blue emission filter; FAD channel: excitation 860 nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.

Whole Brain Imaging with Serial Two-Photon Tomography.

Imaging entire mouse brains at submicron resolution has historically been a challenging undertaking and largely confined to the province of dedicated atlasing initiatives. This has limited systematic investigations into important areas of neuroscience, such as neural circuits, brain mapping and neurodegeneration. In this article, we describe in detail Serial Two-Photon (STP) tomography, a robust, reliable method for imaging entire brains with histological detail. We provide examples of how the basic methodology can be extended to other imaging modalities, such as Optical Coherence Tomography (OCT), in order to provide unique contrast mechanisms. Furthermore, we provide a survey of the research that STP tomography has enabled in the field of neuroscience, provide examples of how this technology enables quantitative whole brain studies, and discuss the current limitations of STP tomography-based approaches.